Methods and processes for non-invasive assessment of genetic variations

Abstract

Provided herein are methods, processes and apparatuses for non-invasive assessment of genetic variations.

1 .- 28 . (canceled) 
     
     
         29 . A computer-implemented method for generating a classification of presence or absence of a sex chromosome aneuploidy for test sample nucleic acid, comprising:
 (a) obtaining counts of sequence reads mapped to portions of a reference genome, which sequence reads are reads of isolated circulating cell-free nucleic acid from the test sample nucleic acid, wherein the test sample nucleic acid is from a pregnant female bearing a fetus;   (b) determining, using a microprocessor, a guanine and cytosine (GC) bias coefficient for the test sample nucleic acid based on a fitted relation between (i) the counts of the sequence reads mapped to each of the portions, and (ii) GC content for each of the portions, wherein the GC bias coefficient is a slope for a linear fitted relation or a curvature estimation for a non-linear fitted relation;   (c) calculating, using a microprocessor, a genomic section level (L i ) for the sections based on the counts of (a), the GC bias coefficient of (b) and a fitted relation, for each of the portions, between (i) the GC bias coefficient for the test sample and (ii) the counts of the sequence reads mapped to each of the portions for the test sample, which calculating generates normalized genomic section levels;   (d) calculating, using a microprocessor, a Z-score from the normalized genomic section levels; and   (e) generating, using a microprocessor, the classification of the presence or absence of the sex chromosome aneuploidy for the test sample nucleic acid according to the Z-score calculated in (c).   
     
     
         30 . The method of  claim 29 , further comprising:
 isolating circulating cell-free nucleic acid from blood of the pregnant female bearing a fetus prior to (a).   
     
     
         31 . The method of  claim 29 , wherein the obtaining counts comprises:
 mapping, using a microprocessor, the sequence reads to portions of a reference genome; and   counting, using a microprocessor, the sequence reads mapped to the portions, which counting generates counts of sequence reads mapped to the portions.   
     
     
         32 . The method of  claim 29 , further comprising sequencing the test sample nucleic acid by a genome-wide massively parallel sequencer that generates sequence reads, which test sample nucleic acid comprises the isolated circulating cell-free nucleic acid. 
     
     
         33 . The method of  claim 32 , wherein the sequencing is at about 1-fold coverage or less. 
     
     
         34 . The method of  claim 29 , wherein the fitted relation of (b) is linear and is determined by a linear regression. 
     
     
         35 . The method of  claim 29 , wherein the GC bias coefficient for each of the multiple samples is the slope of a linear relation between (i) the counts of the sequence reads mapped to each of the portions of the reference genome for each of the samples, and (ii) the GC content for each of the portions for each of the samples. 
     
     
         36 . The method of  claim 35 , wherein the fitted relation in (b) is linear and the normalized genomic level (L i ) is determined for each of the portions of the reference genome according to Equation α:
     L   i =( m   i   −G   i   S ) I   −1   Equation α
 
 wherein G i  is the GC bias coefficient, I is the intercept of the fitted relation in (c), S is the slope of the relation in (c), m i  is measured counts mapped to each of the portions of the reference genome and i is a sample. 
 
     
     
         37 . The method of  claim 29 , further comprising applying a secondary normalization to the genomic section level calculated in (c). 
     
     
         38 . The method of  claim 37 , wherein the secondary normalization comprises GC normalization. 
     
     
         39 . The method of  claim 29 , further comprising determining a chromosome X elevation and a chromosome Y elevation from a plurality of genomic section levels calculated in (c). 
     
     
         40 . The method of  claim 39 , further comprising plotting the chromosome X elevation, or derivative thereof, versus the chromosome Y elevation, or derivative thereof, on a two-dimensional graph, thereby generating a plot position. 
     
     
         41 . The method of  claim 40 , further comprising determining sex chromosome karyotype for the fetus according to the plot position. 
     
     
         42 . The method of  claim 29 , further comprising:
 calculating a measure of error for the counts of sequence reads mapped to some or all of the portions of the reference genome; and   removing or weighting the counts of sequence reads for certain portions of the reference genome according to a threshold of the measure of error, mappability, repeatability, genomic portion-specific t-statistic or combination thereof.   
     
     
         43 . The method of  claim 42 , wherein the threshold is selected according to a standard deviation gap between a first genomic section level and a second genomic section level of 3.5 or greater. 
     
     
         44 . The method of  claim 42 , wherein the measure of error is an R factor and the sequence read count for a portion of the reference genome having an R factor of 7% or greater is removed. 
     
     
         45 . The method of  claim 29 , wherein the portions of the reference genome are in one or more sex chromosomes. 
     
     
         46 . The method of  claim 45 , wherein the number of portions of the reference genome is 20 or more portions for chromosome Y. 
     
     
         47 . The method of  claim 46 , wherein the portions for chromosome Y are chosen from among chrY_125, chrY_169, chrY_170, chrY_171, chrY_172, chrY_182, chrY_183, chrY_184, chrY_186, chrY_187, chrY_192, chrY_417, chrY_448, chrY_449, chrY_473, chrY_480, chrY_481, chrY_485, chrY_491, chrY_502, chrY_519, chrY_535, chrY_559, chrY_1176, chrY_1177, and chrY_1178. 
     
     
         48 . The method of  claim 47 , wherein the portions for chromosome Y comprise one or more of chrY_1176, chrY_1177, and chrY_1178. 
     
     
         49 . The method of  claim 47 , wherein the portions for chromosome Y do not comprise one or more of chrY_1176, chrY_1177, and chrY_1178. 
     
     
         50 . The method of  claim 47 , further comprising: comparing genomic section levels, or derivatives thereof, for one or more of chrY_1176, chrY_1177, and chrY_1178, to genomic section levels, or derivatives thereof, for one or more of chrY_125, chrY_169, chrY_170, chrY_171, chrY_172, chrY_182, chrY_183, chrY_184, chrY_186, chrY_187, chrY_192, chrY_417, chrY_448, chrY_449, chrY_473, chrY_480, chrY_481, chrY_485, chrY_491, chrY_502, chrY_519, chrY_535 and chrY_559, thereby generating a comparison. 
     
     
         51 . The method of  claim 50 , wherein sequence read counts for one or more of chrY_1176, chrY_1177, and chrY_1178 are removed or replaced according to the comparison. 
     
     
         52 . The method of  claim 29 , wherein a subset of portions in chromosome X in the reference genome are utilized for generating the classification, and
 wherein the subset comprises about 2350 or more portions for chromosome X.   
     
     
         53 . The method of  claim 29 , wherein the reference genome is from a male subject. 
     
     
         54 . The method of  claim 29 , wherein the reference genome is from a female subject. 
     
     
         55 . The method of  claim 29 , wherein each portion of the reference genome comprises a nucleotide sequence of a predetermined length. 
     
     
         56 . The method of  claim 55 , wherein the predetermined length is 50 kilobases. 
     
     
         57 . The method of  claim 29 , further comprising generating and transmitting a laboratory prenatal test report comprising the classification of the presence or absence of the sex chromosome aneuploidy for the test sample. 
     
     
         58 . The method of  claim 57 , wherein the report comprises one or more of a sensitivity, specificity and confidence interval for the classification. 
     
     
         59 . The method of  claim 58 , wherein the sensitivity is 95% or greater and the specificity is 99% or greater. 
     
     
         60 . The method of  claim 29 , further comprising monitoring health of the fetus and pregnant female from whom the test sample was obtained based on the classification for the test sample. 
     
     
         61 . The method of  claim 29 , wherein the sex chromosome aneuploidy is chosen from XXX, X, XXY and XYY. 
     
     
         62 . The method of  claim 29 , wherein the nucleic acid is from blood plasma or blood serum. 
     
     
         63 . The method of  claim 29 , further comprising: performing a medical procedure comprising amniocentesis or chorionic villus sampling for the pregnant female.